Outline

The replication complexes of mammalian positive-stranded RNA viruses associate with host cell membrane compartments, which are often modified to become a scaffold for viral RNA synthesis. This early stage of virus replication is considered a potential target for antiviral therapy. In the case of nidoviruses (the arterivirus EAV and the coronavirus MHV) the replication complex has been reported to localize to unusual double-membrane vesicles (DMVs), which appear to be derived from the endoplasmic reticulum. Using recently produced antisera recognizing various SARS-CoV nonstructural

proteins (nsps), we are currently investigating the formation and origin of the SARS-CoV replication complex by using (immuno)electron microscopy. In the more extensively studied arterivirus model, we have previously documented the crucial role of nsp2 and nsp3. Both proteins have significant hydrophobic domains and associate with membranes upon their individual expression from an alphavirus expression vector. We have now used site-directed mutagenesis to investigate the role of nsp2 and nsp3 in DMV formation.

A hallmark of nidovirus RNA synthesis is the production of subgenomic (sg) mRNAs that form a 3'-coterminal nested set with the viral genome, whereas they also contain a common 5' "leader sequence" that is derived from the 5'-proximal region of the genome. The fusion of the "leader" and "body" parts of sg RNAs involves discontinuous RNA synthesis during which the nascent RNA strand is transferred (most likely during minus strand synthesis) from one site in the genomic template to another. Short conserved transcription-regulating sequences at these template sites (leader TRS and body TRSs) play an essential role in this strand transfer mechanism, which resembles similarity-assisted RNA recombination. However, the TRSs are not the only determinant of sg RNA synthesis, and using the arterivirus prototype EAV we are currently investigating the role of specific RNA signals and viral nonstructural proteins (nsps). A role in subgenomic RNA synthesis is emerging for EAV nsps 1, 10, and 11, the latter two being helicase and (putative) endonuclease enzymes that are conserved in coronaviruses. Furthermore, an RNA secondary structure model of the 5'-proximal region of the EAV genome (supported by phylogenetic, mutagenesis and structure probing studies) revealed that the leader TRS is presented in the loop of a hairpin structure. Evidence supporting the importance of this leader TRS hairpin (LTH) for subgenomic RNA synthesis was obtained. A similar LTH was predicted for other arteriviruses and coronaviruses, suggesting that this hairpin may be a common feature of nidovirus sg mRNA synthesis. Using the EAV model, we investigated how body TRS activity affects the function of neighboring body TRSs. Our data underline the complex and multifactorial nature of the regulation of nidovirus sg mRNA abundance.